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Microbiology - Pouring Plates

Jeff Lawrence

1. For rich media, weigh out appropriate ingredients and place into a flask. Add water until appropriate volume. Use a flask at least 2 times larger than the media volume.

2. For minimal medium, make separate 2X agar and 2X salt flasks. For example, for 1 liter of E plates, make 500 mLs of 2X E salts, and 500 mLs of 2 X agar (1.5 g per 500 mLs). Use at least one flask large enough to mix the media together after autoclaving.

3. Cap with a polypropylene beaker and autoclave small batches 20 min; autoclave large volumes 30-40 min. Also autoclave a 1 L flask for pouring large volume of media.

4. Cool to 55° on a bench or in a water bath (37° initial temp). For minimal media, combine the 2X agar with the 2X salt at this point. If they are combined too early, the agar will carmelize in the high salt solution.

5. Add antibiotics and other additions if necessary.

6. For large volumes, transfer a portion to the 1 liter pouring flask. Flame the top of the media to remove the bubbles.

7. Pour each plate to 1/2 volume, about 25-30 mL. This will yield about 35 plates per liter of media. If you are getting 25 plates or fewer, the plates are too thick. If you are getting more than 40 plates, they are too thin. Thin plates dry out quickly and are difficult to use. Thick plates waste media and are hard to print.

8. Pour the bottom plate of a stack first, holding up the remainder of the stack with your free hand. Cap the plate and pour the next one until the stack of 20 is completed. Label the top plate of each stack.

8. Allow plates to cool until solid. The cooler the media is upon pouring, the faster they will solidify.

9. Transfer plates to the 37° incubator and incubate overnight until dry. Fresh, wet plates do not absorb liquids well and are too wet to use for replica plating.